Compositions and method for reducing free fatty acid and cholesterol in the blood of mammals with 3,5 - disubstituted isoxazoles



United States Patent COMPOSITIONS AND METHOD FOR REDUCING FREE FATTY ACID AND CHOLESTEROL IN THE BLOOD OF MAMMALS WITH 3,5 DISUBSTI- TUTED ISOXAZOLES John B." Wright, Kalamazoo, Mich., assignor to The Upjohn ,Company, Kalamazoo, Mich., a corporation of Delaware No Drawing. Continuation-impart of applications Ser. No.

167,911, Jan. 22, 1962, and Ser. No. 237,329, Nov. 13,-

1962.-This application Mar. 4, 1965, Ser. No. 437,265

. Int. Cl. A61k 27/00 US. Cl. 424-272 6 Claims ABSTRACT OF THE DISCLOSURE Reduction of free fatty acids and cholesterol in the blood with 3,5-disubstituted isoxazoles.

wherein R and R are alkyl, carboxyl, alkyl carboxylate, carbamoyl, and monoand dialkyl substituted carbamoyl, and (2) physiologically acceptable salts of the monoor dicarboxyisoxazoles above, including alkali metal salts (such as sodium and potassium), alkaline earth metal salts (such as aluminum, calcium and magnesium), ammonium salts, and amine salts (such as N,N"-dibenzylethylenediamine, procaine and diethanolamine). The alkyl groups contain 1 through 4 carbon atoms.

These compounds have been unexpectedly found to exhibit many times the oral hypoglycemic activity of tolbutamide in standard test animals, e.g., rats. The pronounced activity of these 3,5-disubstituted isoxazoles, coupled'with their low toxicity, makes pharmaceutical compositions containing these compounds useful in reducing the blood sugar content of mammals, and in particular for administration by the preferred oral route in the management of human diabetes.

It has also been discovered that representative compounds of the group here disclosed are effective in reducing the free fatty acids, cholesterol and triglycerides content of blood in mammals. These activities, demonstrated at acceptable therapeutic ratios, make the compounds herein useful as compositions in treating such conditions as atherosclerosis, arteriosclerosis, blood-clotting defects due to lipemia, and other clinical entities in which the underlying etiology is associated with lipid imbalance or hyperlipidemia.

In utilizing the compositions and practicing the method of this invention, the exact schedule of administration in humans and animals is determined individually according to the subjects age, weight, response to the medication and nature and severity of the condition being treated. In each of the disclosed applications, for example, from about 0.5 to about 500 mg. of 3,5-disubstituted isoxazole can be administered orally as a single dose one to four 3,524,001 Patented Aug. 11, 1970 ice times daily, single adult oral doses of from about 2 to about 100 mg. being preferred.

In addition to 3,5-disubstituted isoxazole as the sole active ingredient, other complementary ingredients can be included in the composition to secure advantageous combinations of properties especially adapted to individual situations in the treatment of the foregoing conditions. Thus, other hypoglycemic agents such as tolbutamide, chlorpropamide, phenformin hydrochloride, mesoxalic acid, insulin, nicotinic acid, and the like can be included in the present formulations in amounts not exceeding and preferably less than those normally employed in single unit doses where such added ingredients are employed alone. Utilizable potassium salts, such as potassium chloride, can be included to offset possible potassium losses during therapy.

Such combinations include also conventional therapeutic amounts or less of other hypocholesteremic or other hypolipidemic agents such as the D-isomer of 3,5,3- triiodothyronine, triiodothyropropionic acid, and throxinelike compounds such as sodium L-thyroxine and sodium D-thryoxine; glucocorticoids such as hydrocortisone, prednisolone, 6a-fiuoroprednisolone and 6a-methylprednisolone; anticoagulants such as heparin, Z-diphenylacetyl- 1,3-indandione polyethylene sulfonate and dicumarol or its derivatives; vitamins such as nicotinic acid, vitamin B ascorbic acid and pyridoxine hydrochloride; estrogens such as estradiol; androgens such as testoterone and 7- methylandrostanes such as 7aor 7fl-methyl-5a-androstane-3fl, 17,8-diol; combinations of estrogens and androgens such as estradiol and testosterone; unsaturated fatty acids or esters such as corn oil and linoleic acid or esters; antibiotics such as neomycin; analgesics such as aspirin; compounds associated with cholesterol synthesis or metabolism such as a-phenylbutyric acid and a-p-bipheny1yl butyric acid; lipotropic agents such as choline and inositol; amino acids such as taurine and glycine; sterols such as sitosterol and other plant sterols; diuretics such as ethoxzolamide and hydrochlorothiazide; anorexigenic agents such as amphetamine; cardiovascular agents (including vasodilators and hypotensive agents), such as chlorisondamine chloride, hexamethonium chloride, and pentaerythritol tetranitrate.

In adapting the active ingredients for use in mammals, including humans, the novel compositions are suitably presented for administration in unit dosage form as tablets,

pills, capsules, powders, wafers, cachets, granules, oral aqueous or oil dispersions, including elixirs, and the like.

For preparing solid compositions such as tablets, the active ingredient is mixed with a conventional non-sugar tableting component such as cornstarch, dicalcium phosphate, terra alba (calcium sulfate), talc, stearic acid, calcium stearate, gums, and functionally similar materials constituting pharmaceutical diluents, granulating agents, lubricants or carriers. The tablets or pills can be laminated or otherwise compounded to provide a dosage form affording the advantage of prolonged or delayed action or of predetermined successive action of the enclosed medication. For example, the tablet or pill can comprise an inner dosage and outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids or mixtures of polymeric acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate phthalate and the like. A particularly advantageous sustained release coating comprises a styrene maleic acid copolymer.

The liquid form in which the novel compositions of this invention can be incorporated include aqueous sugarfree solutions or suspensions, emulsions or suspensions with edible oils such as cottoneed oil, sesame oil, coconut oil, peanut oil and the like, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include the synthetic and natural gums such as tragacanth, acacia, alginate, dextran, methylcellulose, polyvinylpyrrolidone, gelatin and the like.

In preparing pharmaceutical compositions of 3,5-dimethylisoxazole, the fact that this compound is a waterinsoluble, volatile liquid must be considered, Appropriate oral liquid dosage forms include conventional sugar-free syrups, elixirs and non-aqueous solutions for use as drops or by the teaspoonful. Suitable non-aqueous vehicles for oral use include the edible oils (e.g., peanut oil, corn oil, cottonseed oil, coconut oil and other vegetable oils), mineral oil, glycerol, propylene glycol, polyethylene glycol 200-600, sorbitol, ethanol, or mixtures of these (e.g., equal parts of peanut oil and propylene glycol). Aqueous vehicles include from about 1 to about 50% aqueous solutions of propylene glycol or ethanol or mixtures of the two comprising together the said percentages. Liquid preparations for intramuscular or subcutaneous injection can be prepared as ethanolic-aqueous, propylene glycol-aqueous or oil solutions (e.g., vegetable oils such as peanut oil) and in repository-type vehicles such as aluminum monostearate-peanut oil gel. In general, liquid formulations range in concentration from about 0.05 to 20-40% 3,5-dimethylisoxazole.

The solid dosage forms of 3,5-dimethylisoxazole require the intermediate preparation of the liquid active ingredient as discrete solid particles which can be employed to build the ultimate dosage form by conventional methods. Alternatively, the 3,5-dimethylisoxazole can be dissolved or dispersed in an edible oil such as a vegetable or mineral oil (in ratios of, for example, about 1:1 to 1:200) and soft elastic capsules containing the oil dispersion or solution prepared for oral use. Triturates of 3,5- dimethylisoxazole can be made using various absorbing powders such as kaolin, magnesium carbonate, bentonite, magnesium oxide, starch, calcium carbonate, tribasic calcium phosphate, magnesium trisilicate and the like. Emulsifying the liquid 3,5-dimethylisoxazole, preferably dissolved in a suitable vegetable oil or mineral oil, to provide small particle sizes and then coating the said particles with a coacervate of one or more hydrophilic colloids, pharmaceutically acceptable copolymers, or mixtures thereof yields, on drying, free-flowing granulations which can be handled essentially as solid particles and formulated as powders, granules, tablets, hard-filled capsules and the like.

Other 3,5-disubstituted isoxazoles as hereinbefore defined are prepared by methods well established in the art, as representatively illustrated in the examples below.

The term unit dosage form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The specification for the novel unit dosage forms of this invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding such an active material for therapeutic use in humans, as disclosed in detail in this specification, these being features of the present invention. Examples of suitable unit dosage forms, as heretofore described, are tablets, capsules, pills, powder packets, wafers, cachets, granules, solutions or suspensions for oral or sterile injectable use, suppositories, and segregated multiples of any of the foregoing, and other forms alluded to herein.

4 The following examples illustrate the best mode contemplated by the inventor for carrying out the invention but are not to be construed as limiting the scope thereof.

EXAMPLE 1 Soft gelatin capsules (3,5-dimethylisoxazole) A batch of 1,000 soft gelatin capsules, each containing 2 mg. of 3,5-dimethylisoxazole in mineral oil, is prepared from the following materials:

Gms. 3,5-dimethylisoxazole 2 Mineral oil, U.S.P. 10

A uniform dispersion of the active ingredient in the mineral oil is prepared and the dispersion filled into soft gelatin capsules by conventional means.

One capsule is given two to four times daily to reduce free fatty acids concentration in the blood.

EXAMPLE 2 Compressed tablets (5-carboxy-3-methylisoxazole) A solution of 3.6 gm. (0.0275 mole) of acetopyruvic acid and 2.0 gm. (0.0285 mole) of hydroxylamine hydrochloride in 60- ml. of water was heated at 88 C. on the steam bath for 3 hours. The solution was cooled to 15 C. and filtered to obtain 1.4 gm. (40%) of material which melted at 183-200 C. (dec.). This material was recrystallized 3 times from acetone to give 300 mg. (9%) of 5-carboxy-3-methylisoxazole, which melted at 209.5- 2l1.5 C.

Analysis.Calcd. for C H NO (percent): C, 47.25; H, 3.97; N, 11.02. Found (percent): C, 47.36; H, 3.76; N, 10.83. Infrared and NMR spectra support the structure.

A lot of 10,000 oral tablets, each containing 10 mg. of S-carboxy-3-methylisoxazole, is prepared from the following ingredients:

The 5-carboxy-3-methylisoxazole and terra alba are mixed well, granulated with 7.5% solution of methylcellulose in water, passed through a No. 8 screen and dried at 120 F. The dried granules are passed through a No. 12 screen, mixed thoroughly with the tale and stearate and compressed into tablets.

One tablet is given three times daily in reducing free fatty acids and cholesterol content of blood.

Inclusion of 2500 gm. of tolbutamide in place of the terra alba in the above formulation gives a combination of 10 mg. of 5-carboxy-3-methylisoxaz0le with 250 mg. of tolbutamide useful in the treatment of adult diabetes at a dose of 1 or 2 tablets three times daily.

Inclusion of 5000 gm. of nicotinic acid in place of the terra alba in the above basic formulation gives a combination of 10 mg. of S-carboxy-3-methylisoxazole with 500 mg. of nicotinic acid useful in lowering cholesterol content of blood at a dose of 1 or 2 tablets three times daily.

EXAMPLE 3 Hard gelatin capsules (3-carboxy-S-methylisoxazole) Twenty grams of acetonyl acetone and 40 gm. of nitric acid were dissolved in 60 ml. of water and heated gently to initiate an exothermic reaction. After boiling for 25 minutes, the reaction mixture was permitted to stand overnight. The mixture was then diluted with water, extracted with ether, concentrated and steam distilled. The aqueous residue was concentrated to a brown oily mass, dried and dissolved in ml. of acetone. Solids were filtered from the acetone solution, which was concentrated and again azole, is prepared from the following materials:

. L Gms. 3-carboxy-S-methylisoxazole, micronized 1000 Potassium chloride 1000 Mineral oil, USP 129.6 Magnesium stearate, powder 162 Talc, USP 162 Corn starch I616 The powdered 3-carboxy-S-methylisoxazole is mixed thoroughly with the rest of the powdered ingredients and mineral oil and then encapsulated.

One capsule four times daily is given in the treatment of human diabetes.

EXAMPLE 4 Oil suspension (S-carbamoyl-3-methylisoxazole) A mixture of 4.3 gm. (0.033 mole) of 5-carboxy-3- methylisoxazole in 25 m1. of thionyl chloride Washeated on the steam bath for 24 hours. The excess thionyl chloride was distilled off and the residue added cautiously to 50 ml. of cold, concentrated ammonium hydroxide. The mixture was stirred in an ice bath for 15 minutes and filtered. Recrystallization from isopropanol and treatment with activated carbon gave 1.7 gm. (41%) of S-carbamoyl-3-methylisoxazole melting at 184188 C.

Analysis.Calcd. for C H N O (percent): C, 47.62; H, 4.80; N, 22.22. Found (percent): C, 47.96; H, 4.79; N, 22.04. Infrared spectrum supports the structure.

An oil suspension for oral use, each 5 ml. containing 150 mg. of 5-carbamoyl-3-methy1isoxazole, is prepared from the following materials:

One teaspoonful (6 m1.) is given twice daily in the treatment of diabetes.

EXAMPLE 5 Injectable. preparation (3,5-diethylisoxazole) An injectable preparation is made from the following ingredients to contain 100 mg. of 3,5-diethylisoxazole per ml.:

Percent (w./v.)

Sodium carboxymethylcellulose (low viscosity) 0.5 Tween 80 (polyoxyethylene sorbitan monooleate) 0.4 Sodium chloride 0.9 Benzyl alcohol 0.9 3,5-diethylisoxazole 10.0

Sterile distilled water, q.s., 100.0 ml.

The previously sterilized 3,5-diethylisoxazole is homogenized with the already mixed and sterilized vehicle.

Administration is on a basis of 1 ml. daily given intramuscularly in the management of adult human diabetes.

Six thousand units of crystalline zinc insulin can be added to the above formulation to give a combination product administrable in 0.5-ml. amounts or less daily.

6 EXAMPLE 6 Capsules (3-dirnethylcarbamoyl-S-methylisoxazole) A mixture of 14.55 gm. (0.1 mole) of S-methylisoxazole-3-carbonyl chloride, 10 gm. of dimethylamine and 50ml. of dry benzene is stirred at 0-5" C. in an ice bath for 24 hours. An equal volume of ether is added, and the precipitated 'dimeth'ylamine hydrochloride is removed by filtration. The filtrate is concentrated to dryness in vacuo.

Recrystallization in the usual manner gives essentially pure 3-dimethylcarbamoyl-S-methylisoxazole.

A lot of 10,000 two-piece hard gelatin capsules for oral use, each containing 250 mg. of 3-dimethylcarbamoyl-5- methylisoxazole, is prepared from the following materials:

"Gms. 3-dimethylcarbamoyl--methylisoxazole 2500 Magnesium stearate, powder 162 Talc, USP 162 The powdered isoxazole is mixed thoroughly with the rest of the ingredients and then encapsulated.

One capsule is given daily in the reduction of triglycerides content of blood.

EXAMPLE 7 In each of the foregoing Examples 1-6 the active ingredient can be replaced by equivalent amounts of other active ingredients heretofore described, e.g., 3,5-dipropyl, diisopropyl or dibutylisoxazole; 3,5-mixed lower alkyl isoxazoles; S-carboxy-3-ethylisoxazole, the corresponding 3-propyl, isopropyl or butylisoxazoles, and alkyl carboxylates and physiologically acceptable salts thereof; 3- carboxy-S-ethylisoxazole, the corresponding 5-propyl, isopropyl or butylisoxazoles and alkyl carboxylates thereof; 5-carbamoyl-3-ethylisoxazole and the corresponding 3-propyl, isopropyl or butylisoxazole; and 3-carbamoyl-5- ethylisoxazole and the corresponding S-propyl, isopropyl or butylisoxazole, and the monoand dialkyl substituted carbamoyl derivatives, all the alkyl groups above containing 1-4 carbon atoms.

What is claimed is:

1. A therapeutic composition comprising: as the primary active ingredient, about 0.5 to about 500 mg. of a compound selected from the group consisting of S-carboxy-3-alkylisoxazole and a physiolog ally acceptable salt thereof, said allcyl group containing 1 through 4 carbon atoms, in combination with a pharmaceutical carrier in unit dosage form.

2. A therapeutic composition comprising: as the primary active ingredient, about 2 to about 200 mg. of 5- carboxy-3-methylisoxazole, in combination with a pharmaceutical carrier in unit dosage form.

3. A method for treating hyperlipidemia in the blood of a mammal comprising: administering to said mammal and effective amount of a 3,5-disubstituted isoxazole selected from the group consisting of a compound of the formula wherein R and R are selected from the group consisting of alkyl, carboxy-l, alkyl carboxylate, carbamoyl, and monoand dialkyl substituted carbamoyl and a physiologically acceptable salt of the said carboxyisoxazole and said alkyl groups containing 1 through 4 carbon atoms, in combination with a pharmaceutical carrier.

4. A method for reducing free fatty acids and cholesterol in the blood of a mammal comprising: administering to said mammal about 0.5 to about 500 mg. of a 3,5-disubstituted isoxazole selected from the group consisting of (1) a compound of the formula wherein R and R are selected from the group consisting of alkyl, carboxyl, alkyl carboxylatc, carbamoyl, and monoand dialkyl substituted carbamoyl, (2) physiologically acceptable salt of the said carboxyisoxazole and said alkyl groups containing 1 through 4 carbon atoms, in combination with a pharmaceutical carrier.

5. A method for reducing free fatty acids and cholesterol in the blood of a mammal comprising: administering to said mammal about 2 to about 200 mg. of 3,5-dimethylisoxazole, in combination with a pharmaceutical carrier.

6. A method for reducing free fatty acids and cholesterol in the blood of a mammal comprising: administering to said mammal about 2 to about 200 mg. of S-carboxy-B-methylisoxazole, in combination with a pharmaceutical carrier.

References Cited UNITED STATES PATENTS 1/1963 Kano et a1. 260'30 7 OTHER REFERENCES Chem. Abst. (I), vol. 45:7107G (1951). Chem. Abst (H), vol. 45:9036C (1951).

ALBERT T. MEYERS, Primary Examiner S. J. FRIEDMAN, Assistant Examiner U.S. Cl. X.R. 

